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We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.

We have labeled proinsulin connecting peptide (C-peptide) with fluorine-18 (t½=109.7 min) in order to perform in vivo biodistribution and pharmacokinetic studies with position emission tomography (PET). This study reports the optimization of the conjugation labeling in the N-terminal with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). In preparative runs N-4-[¹⁸F]fluorobenzoyl-C-peptide ([¹⁸F]FB-C-peptide) was produced in 8–12% decay-corrected yields, counted from resolubilized [¹⁸F]F⁻, in less than 5 h. The specific radioactivity of [¹⁸F]FB-C-peptide, determined using ELISA for one of the preparations, was around 70 GBq/μmol at end of synthesis. Copyright © 2001 John Wiley & Sons, Ltd.

Hydrogen/deuterium (H/D) exchange measurements in low and moderate concentrations of GuHCl were conducted on the side chain HNatoms of the seven tryptophans of pseudo wild‐type human carbonic anhydrase II. Tryptophans 5, 16 and 245, situated in or close to the N‐terminal domain were found to have little protection against exchange. The H/D exchange results for Trp‐123, Trp‐192 and Trp‐209 showed that a previously identified molten globule and the native state gave a similar protection against exchange. Global unfolding of the protein is necessary for the efficient exchange at Trp‐97, which is located in the central part of the β‐sheet.

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity ( t12=9 min). Kinetic data from ‘double-jump’ measurements indicate that proline isomerization might be rate determining, in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics; the presence of isomerase during reactivation lowers the half-time or the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis-peptidyl-Pro bonds at Pro 30and Pro 202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) wore constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPlase the P202N mutant behaved exactly like the unmutaled enzyme, Thus, cis-transisomerization of the Pro 202cis-peptidyl bond is not rate determining in the reactivation process, The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.

Streptococcal protein G (SPG) is a cell surface receptor protein with a multiple domain structure containing tandem repeats of serum albumin-binding domains (ABD) and immunoglobulin-binding domains (IgBD). In this paper, we have analysed the fold of ABD. Far-UV circular dichroism analysis of ABD indicates high helical content (56%). Based on an analysis of nuclear magnetic resonance 13C secondary chemical shifts, sequential and short-range NOEs, and a few key nuclear Overhauser effects, we conclude that the ABD is a three-helix bundle. The structure of the ABD is, thus, quite different from the IgBD of protein G [Gronenborn, A.M. et al. (1991) Science 253, 657–661]. This strongly suggests that the ABD and the IgBD of SPG have evolved independently from each other. However, the fold of ABD is similar to that of the IgBD of staphylococcal protein A, possibly indicating a common evolutionary ancestor, despite the lack of sequence homology.

The finite-element method and the discrete-block method have been used to study the defor-mational behaviour of a jointed rock mass subjected to simple shear displacement. Blocks in models with large sizes of block make contact at few points which cause large openings of joints and high stress concentrations in the blocks and surrounding joints. The introduction of more joints into the models evens out the forces, reduces the stresses and causes a gentle type of deformation. From this study, we draw the practical conclusion that joints in rock plinths are eligible for the storage of radio-active waste, if we take into account the effect of simple shear displacement from faulting.

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t½=9 min). Kinetic data from ‘double‐jump’ measurements indicate that proline isomerization might be rate determining, in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics; the presence of isomerase during reactivation lowers the half‐time or the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis‐peptidyl‐Pro bonds at Pro30and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) wore constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPlase the P202N mutant behaved exactly like the unmutaled enzyme, Thus, cis‐transisomerization of the Pro202cis‐peptidyl bond is not rate determining in the reactivation process, The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.

We show that coexpression of a specific binding protein in Escherichia coli can significantly improve the relative yields of correctly folded human insulin-like growth factor I (IGF-I). A glutathione redox buffer was used during growth to allow formation and breakage of disulfide bonds in the periplasm of the bacterial host. Both the binding protein and the peptide hormone were produced as affinity fusions, which allowed purification of the in vivo formed heterodimer by alternative affinity purification methods. The use of affinity-assisted in vivo folding has general implications for expression, folding, and purification of recombinant proteins.

Streptococcal protein G (SPG) is a cell surface receptor protein with a multiple domain structure containing tandem repeats of serum albumin‐binding domains (ABD) and immunoglobulin‐binding domains (IgBD). In this paper, we have analysed the fold of ABD. Far‐UV circular dichroism analysis of ABD indicates high helical content (56%). Based on an analysis of nuclear magnetic resonance 13C secondary chemical shifts, sequential and short‐range NOEs, and a few key nuclear Overhauser effects, we conclude that the ABD is a three‐helix bundle. The structure of the ABD is, thus, quite different from the IgBD of protein G [Gronenborn, A.M. et al. (1991) Science 253, 657–661]. This strongly suggests that the ABD and the IgBD of SPG have evolved independently from each other. However, the fold of ABD is similar to that of the IgBD of staphylococcal protein A, possibly indicating a common evolutionary ancestor, despite the lack of sequence homology.